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SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES

Respiratory syncytial virus (RSV) is the leading cause for childhood hospitalization and respiratory distress, being recognized as a major health and economic burden worldwide. RSV can exploit host immunity and cause a strong inflammatory response that leads to lung damage and virus dissemination. Unfortunately, the immune response elicited by RSV normally fails to protect against subsequent exposures to the virus. Despite intense research during the 50 years after the discovery of RSV, scientists are just beginning to understand the mechanisms contributing to pathology and to the inadequate immune response shown by susceptible individuals. Here, we discuss some of the most important advances made in this field that could lead to the development of new prophylactic tools. Copyright © 2012 John Wiley & Sons, Ltd.


Abbreviations used
BCG

Bacille Calmette Guérin

DC

dendritic cell

FI-RSV

formalin-inactivated RSV preparation

IS

immunological synapses

mDCs

myeloid dendritic cells

pDCs

plasmacytoid dendritic cells

Tregs

regulatory T cells

INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants and young children in the world, as well of infant hospitalization due to respiratory disease. In the USA alone, RSV causes approximately 125 000 annual hospitalizations [1]. Because of the high infectivity of the virus, nearly 70% of children are infected by the age of 1 year, whereas almost 100% have been exposed to the virus during their second year of life. Importantly, about 36% of infants under 2 years have suffered at least two infections [2, 3]. Although infection rates for RSV are similar throughout the world, severe pathology predominantly occurs in infants displaying risk factors, such as incomplete airway development due to premature birth, pulmonary hypertension, airway hyper-reactivity, congenital heart disease and immunosuppression [4]. Furthermore, RSV-associated acute lower respiratory infection and RSV-associated fatality ratios are higher in infants residing in developing countries [5].

Respiratory syncytial virus belongs to the Paramyxoviridae family, which also includes measles, parainfluenza 3, Hendra and Nipah viruses. Together with the recently identified human metapneumovirus, RSV is more specifically classified into the subfamily Pneumovirinae [6]. RSV is an enveloped virus with 10 genes distributed along 15.2 kilobases of negative-stranded RNA that encode 11 proteins (Figure 1). Eight of the RSV proteins are known to be structural and so present in the virion particle (Figure 1). The two non-structural proteins, NS1 and NS2, are expressed only during cell infection and are not packaged into the virion. Whether the M2 gene product, M2-2, is packaged within the virion remains to be determined.

image

Figure 1. Structure of the respiratory syncytial virus (RSV) virion. RSV is an enveloped virus with eight known structural proteins. The fusion protein (F), the attachment glycoprotein (G) and the small hydrophobic protein (SH) are located on the surface of the virion. The ssRNA genome and the remaining viral structural proteins, which are the matrix (M), the nucleocapsid (N), the RNA-dependent RNA polymerase (L), the phosphoprotein (P) and the M2 gene product M2-1, reside inside the envelope. The exact location of the M2 gene product M2-2 is currently unknown

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Despite extensive research efforts, no vaccines are currently available that are capable of inducing long-lasting protective immunity against this virus. Nevertheless, although a widely used prophylactic strategy based on a humanized neutralizing antibody has been shown to be effective at preventing RSV-induced damage, its high cost has limited the use of this treatment only for high-risk groups, such as preterm infants (<37 weeks of gestational age) and infants suffering from cardiovascular diseases and immunosuppression. Further, the only available antiviral drug for treating RSV replication, ribavirin, has shown questionable cost-effectiveness [7]. Thus, it is essential to generate new treatments or ideally a vaccine for RSV that could be affordable by public health systems worldwide. Such an effort will require a better understanding of the pathology caused by RSV and its effects on the host immune system.

In this review, we discuss the latest findings in the field of RSV infection, pathology and virulence that underscore the complexity of the immune response triggered by this pathogen. Furthermore, we describe new experimental strategies to prevent the pathology induced by the exposure to RSV early in life.

RESPIRATORY SYNCYTIAL VIRUS IMMUNITY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES

Protective immunity to respiratory syncytial virus and respiratory syncytial virus-induced immunopathogenesis

In most healthy adults, symptoms elicited after RSV infection manifest as rhinitis, and the course of the disease resembles a common cold. However, RSV can cause severe inflammation of the respiratory tract in susceptible young children, the elderly and immunosuppressed individuals [5, 8, 9]. Further, in some susceptible patients, RSV may also produce damage in other tissues besides the lungs, such as the brain, heart and liver [10-12]. It has been estimated that between 0.5% and 3% of children younger than 2 years manifest severe respiratory symptoms after infection with RSV. Considering that RSV infects ~100% of infants by age 2 years, this apparently small percentage becomes very significant [2, 3]. Similar to other respiratory viruses, RSV can repeatedly re-infect the host, with a significant percentage of children re-infected during their second year of life [2, 3, 13]. However, in most cases the severity of the pathology produced by RSV is gradually reduced after re-infection, diminishing significantly with the age of exposure [2, 3]. Although numerous studies have shown an inverse correlation between the presence of neutralizing antibodies and re-infection [14, 15], several other reports suggest that antibodies developed against RSV during natural infection contribute poorly to protection [16, 17]. Lack of antibody-mediated protection could be due to the fact that RSV-blocking antibodies are usually elicited at very low frequencies after infection, requiring in some cases decades of repetitive exposure to the virus for their acquisition [16-18]. A possible explanation for this phenomenon is the poor immunogenicity of viral protein domains required for virulence. Additionally, other viral proteins are produced in large amounts, such as the secreted form of the G attachment glycoprotein, which could work as an antibody decoy promoting the production of ineffective antibodies to irrelevant viral epitopes (Figure 1) [19]. Thus, because of the absence of effective neutralizing antibodies, it is likely that RSV infection is mainly cleared by IFN-γ secreting CD8+ and CD4+ T cells that promote virus clearance either directly by destroying infected cells or indirectly by limiting inflammation in the lungs [20-25]. Indeed, healthy individuals display T-cell activation levels that are sufficient to mediate viral clearance from the lungs, which promotes low levels of inflammation and hence reduced tissue pathology. The requirement of T-cell immunity for RSV clearance is underscored by the observation that HIV-1-infected patients display increased RSV titres for up to 199 days and mice depleted of CD4+ and CD8+ T cells display prolonged RSV replication [25, 26]. A recent report suggests that the ratio of CD4+/CD8+ RSV-specific T cells may be key to defining the outcome of infection, with higher CD4+/CD8+ memory T cell ratios prevailing in the blood of healthy adults, as compared with those suffering primary RSV infection [27]. Such a finding may be useful in the future to predict disease outcome and assess the potential of experimental vaccines in animal models [28].

Unlike healthy adults, more susceptible hosts can display several harmful immune responses after RSV infection, such as increased inflammation in the lungs, locally impaired or damaging CD8+ T-cell responses and detrimental polarization of CD4+ T cells [24, 29-32]. All these processes are likely to be driven both by viral determinants and host-cell mediators in response to RSV [22, 33]. Recent studies in mice suggest that RSV infection can desensitize alveolar macrophages to TLR ligands for several months after viral resolution [34]. This process could imprint the affected lungs with a phenotype that promotes airway pathology to subsequent viral or bacterial infections [34]. Furthermore, RSV-infected human epithelial cells have been shown to upregulate the expression of inhibitory surface molecules, such as PD-L1 that dampens T-cell activation [35]. Thus, RSV may increase pathology by remodelling the airways [36].

Respiratory syncytial virus interferes with T-cell function

Although RSV antigens are presented and accessible to T cells during infection, as shown by the identification of immunogenic peptides for several RSV proteins in mice and humans, several studies suggest that T-cell function may be specifically impaired in the respiratory tract of those individuals suffering pathology during RSV infection [31, 32, 37, 38]. This notion is supported by the observation that RSV-specific T cells remain functional in the periphery after viral infection, a phenomenon also seen for other respiratory viruses [31, 37, 38]. These findings have led to the concept that impaired antiviral T-cell immune responses in the lungs may be associated with both the tissue itself and the infecting pathogen. Despite this, CD8+ T cells in the lungs of RSV-infected individuals show reduced intracellular granzyme B content, limited secretion of IFN-γ and failure to upregulate perforin expression upon antigen stimulation [31, 38]. All these immune features are required for the destruction of virus-infected cells. It is noteworthy that production of IFN-γ by CD8+ T cells confronted with RSV can be restored by adding exogenous IL-2, a phenomenon also observed for anergic T cells [31]. Consistent with this notion, in vivo IL-2 supplementation can significantly improve the effector function of lung CD8+ T cells recognizing RSV in infected animals [32]. These studies indicate that cytotoxic T cells recognizing RSV display an altered phenotype at the lungs and do not secrete key cytokines, such as IL-2 and IFN-γ, which are required for the activation and full display of antiviral T-cell activity. Although it is tempting to relate reduced T-cell activation by RSV-infected dendritic cells (DCs) with subsequent host re-infections, this concept was recently challenged by respiratory viruses that block T-cell activation without necessarily re-infecting their hosts more frequently [13].

Other studies have shown that, despite controlling virus replication, RSV-induced T cells contribute to pathology because disease severity can be significantly reduced by depleting mice of these cells [25]. Consistent with this notion is the observation that RSV infection in neonate and adult mice can induce virus-specific CD4+ and CD8+ T cells, which are fully activated during infection yet display detrimental phenotypes that enhance disease severity during primary and secondary infection [29, 39, 40].

These observations suggest that RSV has evolved molecular mechanisms to dampen the antiviral T-cell immune response and also to promote in certain cases the induction of unwanted pro-inflammatory T cells.

Respiratory syncytial virus impairs dendritic cell function to modulate T-cell activation

Pathogens can avoid T-cell immunity by dampening the function of DCs [41, 42], which are professional antigen-presenting cells that sense pathogens through the engagement of pattern recognition receptors. Pathogen encounter generally leads to DC maturation and the secretion of immune-stimulating cytokines that contribute to the activation and polarization of T cells [43-45]. Upon exposure to RSV, most human and murine DCs display abortive levels of viral RNA synthesis, and only a fraction of cells become readily infected (Figure 2) [46-50]. Consistently, only negligible numbers of viral particles can be recovered from the supernatants of infected DCs. Thus, evidence suggests that these cells may be exploited by RSV to manipulate immunity rather than for viral replication. In fact, although DCs can recognize RSV molecular patterns via a variety of receptors, such as TLRs and RIG-1 [43, 44], human and murine DCs undergo only weak maturation after virus encounter. Thus, only a modest upregulation of surface activation markers, such as CD40, CD80, CD86 and MHC (Figure 2) [46-49, 51], is observed for DCs after RSV challenge. Nevertheless, DCs respond to the virus by secreting polarizing cytokines, such as IL-6 and IL-10, which can promote T-cell differentiation into phenotypes that are poorly effective at clearing the virus (Figure 2) [47, 51]. Therefore, RSV seems to have developed molecular strategies to interfere with the function of DCs. Accumulating data suggest that in vitro infected human and murine DCs fail to efficiently prime T cells [47-49, 52], probably because of the reduced capacity of RSV-infected DCs to secrete activating cytokines, such as IL-12, required to induce CD4+ T cells capable of driving the expansion of cytotoxic and memory CD8+ T cells (Figure 2) [22, 38, 46, 47, 49, 53, 54]. However, the identification of host and viral molecular determinants that account for the altered response shown by DCs to RSV infection remains elusive. Along these lines, a recent report showed that neonate and adult human DCs secrete different cytokine patterns in response to RSV, especially for the levels of TGF-β produced [55]. Cord blood-derived DCs secreted significantly more TGF-β1 in response to RSV infection than did DCs obtained from adult blood [55]. Furthermore, contrastingly different cytokine profiles were obtained in the co-cultures of these RSV-infected DCs with autologous T cells. Whereas co-cultures with adult DCs contained IL-2, IL-12, IFN-γ and TNF-α, those with cord DCs contained IL-1β, IL-4, IL-6 and IL-17 [55]. This differential response of neonatal and adult DCs to RSV could contribute to increased infant susceptibility to developing inadequate virus-specific T-cell immunity and lung pathology. Consistent with this notion, neutralization of IL-17 in RSV-infected mice was recently shown to significantly reduce the production of mucus in the airways and decrease viral loads in the lungs [56]. Furthermore, IL-17 neutralization led to an increase in the number of RSV-specific CD8+ T cells, thereby reducing the production of Th2 cytokines in RSV-exacerbated allergic mice. In another study, IL-4−/− mice displayed reduced peribronchial lymphocytic inflammation as well as increased levels of the Th1 cytokine IFN-γ [57]. RSV infection leads to a sustained increase in the lung recruitment of both myeloid (mDCs) and plasmacytoid DCs (pDCs) in mice and humans. Further, as a result of infection, migration of these cells to the lymph nodes also takes place [58-62].

image

Figure 2. Respiratory syncytial virus (RSV) interferes with DC and T-cell function. (1) RSV can infect DCs, as shown by the surface expression of viral encoded proteins, such as the fusion F protein and intracellular expression of viral RNA (nucleocapsid gene). (2) Upon infection with RSV, DCs mature as a result of the engagement of surface and internal pathogen recognition receptors, such as TLRs, lectins and RIG-I by viral determinants. (3) RSV-infected DCs secrete cytokines that can either promote CD4+ T-cell differentiation into Th2 phenotypes (e.g. IL-10 and IL-6) or inhibit their function (e.g. IFN-λ and IFN-α) [49, 53]. Furthermore, infection in neonates may promote the generation of detrimental CD8+ T cells. Additionally, DCs can secrete chemokines, such as CXCL10 and CCL2, that modulate immune cell migration. (4) RSV can impair DC–T cell interaction by interfering with immunological synapse assembly (detailed in Figure 3). (5) The soluble form of the RSV G glycoprotein can interfere with T-cell differentiation and migration by interacting with CX3CR1 receptors expressed on the surface of T cells. (6) Few reports have provided evidence for direct interaction between RSV and T cells at their surface, which has been described to interfere with T-cell cytoskeleton organization in response to activating stimuli. (7) RSV can impair T-cell activation and function in the lungs by reducing IFN-γ secretion. (8) T cells activated in the context of an RSV infection can display Th2 phenotypes and secrete characteristic inflammatory cytokines

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Plasmacytoid DCs accumulating in the lungs of RSV-infected mice secrete IFN-α and other type-I IFNs in response to the virus and are thought to contribute to controlling RSV-mediated lung pathology [59, 63, 64]. On the other hand, DCs subtypes different from plasmacytoid, such as monocyte-derived human DCs, fail to secrete these cytokines in high amounts in response to RSV, which would partly reduce DC maturation [65]. Previous experiments performed on human epithelial cell lines have reported that NS1/2 can interfere with STAT-2 function to modulate NF-κB and IRF-3 activity, leading to altered type-I IFN signalling [66, 67]. A recent report showed that RSV can also interfere with STAT-1 and STAT-2 signalling in murine bone-marrow derived DCs [68]. In fact, NS1 could negatively modulate the capacity of human DCs to activate both CD4+ and CD8+ T cells by decreasing the proliferation of cytotoxic CD8+ T cells that migrate to the lungs and reducing the activation and proliferation of CD4+ Th17 cells with potential antiviral effects [39]. Furthermore, NS1 was seen to promote the capacity of DCs to activate IL-4-secreting CD4+ T cells while reducing the proliferation of total CD4+ T cells. Strikingly, nearly all these effects were shown to be independent of type-I IFN signalling.

Blockade of the chemokine CCL20 during RSV infection in mice reduced the frequency of mDCs in the lungs but did not alter pDC numbers or the recruitment of T cells [69]. Such a treatment ultimately led to reduced lung pathology and an enhanced Th1 effector response against RSV [69]. Similar data were obtained in CCR6−/− animals, which support the notion that pDCs contribute positively to RSV clearance and that CCL20 may enhance this process [59, 69].

Respiratory syncytial virus interferes with dendritic cell–T cell synapse

Another mechanism by which RSV may alter T-cell activation and function is to impair the assembly of productive immunological synapses (IS) between RSV-infected DCs and antigen-specific CD4+ T cells (Figure 3(A)) [47, 70, 71]. Thus, by interfering with IS assembly, RSV could render DCs unable to productively prime T cells, even in the presence of cognate peptide-MHC (Figure 3(B)) [47]. We have recently observed that infected murine DCs show impaired IS formation and fail to induce activation of T cells (Figures 2 and 3) [47]. Under our experimental settings, inhibition of T-cell activation required cell–cell contact and did not seem to depend on the secretion of soluble mediators by infected DCs, as it has been suggested by other studies (Figure 2) [47, 49, 53]. Therefore, inhibition of immunological synapse assembly by RSV is likely to involve molecules expressed by DCs during the infective cycle which are recruited to the DC–T cell interface (Figure 3(B)) [47]. This possibility is consistent with a study showing that human epithelial cells expressing the RSV fusion (F) protein inhibit T-cell activation [72], although this phenomenon has not been described yet for DCs. Further research is required to analyse the mechanisms responsible for the RSV inhibition of DC–T cell synapse assembly, as well as the biological function of other RSV proteins and their contribution to modulating the capacity of DCs to prime T cells.

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Figure 3. Respiratory syncytial virus (RSV) impairs the establishment of the immunological synapse between DCs and T cells. (A) DC–T cell activating immunological synapses (IS) are characterized by cell–cell interfaces containing a peripheral ring harbouring adhesion molecules and an inner ring enriched in TCR–pMHC complexes, co-stimulatory molecules and activating cytokine receptors. Upon engagement with their ligands, these molecules can induce intracellular activating signals that accumulate and lead to T-cell activation and differentiation. (B) However, RSV-infected DCs express viral proteins that may be targeted to the DC–T cell interface, either intracellularly or at the cell surface. Such proteins could impair the localization and/or function of host activating molecules that are required for T-cell activation during IS assembly, progress and termination. In addition, RSV proteins may promote the recruitment of host molecules at the IS that inhibit T-cell activation. Activating and inhibitory cytokines secreted by RSV-infected DCs are likely to further modulate the signalling events taking place within T cells that are required for an adequate cell activation and differentiation. Taken together, the outcome of T-cell stimulation will depend on the integration of both activating and inhibitory stimuli provided by virus-infected DCs

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Additional factors participating during antigen presentation, such as the quantity of surface cognate peptide-MHC, the length of DC–T cell interactions and cytokines surrounding the DC–T cell environment can also influence the outcome of synapse assembly and efficiency and ultimately define the effector phenotype of the responding T cells [73-76]. Thus, the integration of activating and inhibitory signalling events in T cells, resulting from distinct stimuli and signalling pathways including TCR–peptide–MHC interaction, co-stimulatory molecules and cytokine receptors, will define the fate of antigen-specific T cells (Figure 3(B)). These observations emphasize that IS formation is a fine-tuned process that could be exploited at multiple levels either directly or indirectly by RSV virulence determinants to interfere with T-cell function. In fact, the diversity of T-cell phenotypes observed after exposure to RSV, such as pro-inflammatory phenotypes, Th2 commitment, partial T-cell activation or anergy, are in line with viral interference of DC–T cell IS (Figure 3(B)).

Another aspect related to the inhibition of T-cell function, although poorly explored, is the direct effect of RSV on these cells. Although viral proteins interact at the surface of T cells, replication of viral RNA is not observed within these cells, suggesting abortive infection [77]. However, providing RSV to human CD4+ T-cell cultures modulates the cytoskeleton and interferes with the activation of these cells by an unknown mechanism (Figure 2) [52]. These findings suggest that RSV could inhibit the function of non-antigen-specific T cells after infection, which is consistent with the observation that RSV suppresses the activation of bystander murine T cells in vivo as well as in vitro [38, 78].

Respiratory syncytial virus interferes with T-cell polarization

Although controversial [13], the severity of RSV infection in humans and animal models has been in part associated with the expansion of CD4+ T cells that display phenotypes that are non-optimal for virus clearance [39, 40, 79-81]. As part of this type of response, T cells recognizing RSV antigens often polarize towards a Th2 phenotype and secrete or promote the secretion of cytokines, such as IL-4, IL-5 and IL-13 [39, 40, 79, 80, 82-84]. These molecules can positively modulate the activation and recruitment of other immune cells, such as eosinophils, neutrophils and monocytes, into the lungs, which produce and secrete pro-inflammatory molecules contributing to pathology [85-87]. Concomitantly, cytokines such as IL-9 and IL-4 can modulate the immune response by dampening the activity of cytotoxic virus-specific CD8+ T cells (Figure 2) [21, 31, 37, 38, 88]. Contrarily, an increase in the number of Th1-polarized RSV-specific T cells in the lungs can reduce RSV replication and decrease T-cell-associated pro-inflammatory cytokines [21, 24]. Studies in animal models have shown that the natural establishment or induction of a balanced Th1 immunity with vaccination can considerably improve the host response to RSV infection [21, 24, 32]. These observations support the idea that pro-inflammatory responses and their associated cytokines are generally related to RSV-induced pathology. On the contrary, a properly balanced Th1/Th2 polarization is required for limiting virus replication in the infected tissue and reducing lung pathology. These findings have been confirmed by data obtained from vaccination and virus challenge studies in animal models for RSV [21, 89-92].

Although CD8+ cytotoxic T cells are usually considered to be advantageous for viral clearance because they correlate positively with protective immunity in mice successfully vaccinated against RSV, some reports suggest that naturally occurring CD8+ T cells specific to the virus may play detrimental roles during primary infection and secondary challenge with the virus [29, 93]. This possibility is supported by the observation that depletion of CD8+ T cells during primary and secondary infections can reduce the severity of RSV-induced disease in infected animals [29]. It is thought that these detrimental virus-specific occurring CD8+ T cells may be secreting damaging pro-inflammatory cytokines that promote lung damage, similar to CD8+ T cells induced upon RSV-enhanced allergic inflammation [94]. However, it is important to point out that pathological CD8+ T cells could be modulated by regulatory CD4+ T cells in such a way to reduce virus-induced illness without affecting viral clearance in the lungs [93, 95].

Because RSV modulates CD4+ T-cell polarization, it was initially thought that the virus might also promote the expansion of regulatory T cells (Tregs) to downmodulate the activity of virus-specific cytotoxic T cells [49]. However, mice depleted of CD25+FoxP3+ regulatory T cells prior to infection display increased levels of pro-inflammatory chemokines and cytokines in the lungs after infection as compared with their non-depleted counterparts [96]. Furthermore, animals depleted of CD4+FOXP3+CD25+ natural Tregs show delayed recovery, enhanced weight loss and increased numbers of activated natural killer cells, which are thought to be beneficial for virus clearance [97]. This effect could be mediated by IL-10 secreted by these cells, which was recently shown to prevent excessive chemokine production and pro-inflammatory cytokines in the lungs after RSV infection [93]. Moreover, in one of these studies, Tregs were shown to differentially modulate the activity of RSV-specific CD8+ T cells to dominant and subdominant viral antigens [96, 98]. These observations suggest that Tregs play a favourable role during RSV infection by downmodulating the production of pro-inflammatory cytokines that could contribute to decreasing lung inflammation and to shaping T-cell immunity against the virus. Whether Tregs participate during RSV infection in humans remains to be determined. However, the idea of expanding Tregs in infected individuals to decrease lung inflammation and promote effective antiviral CD8+ T-cell function seems to have some promising prospect.

Respiratory syncytial virus interferes with T-cell migration

In addition to modulating the polarization of T cells towards pro-inflammatory phenotypes, the RSV G glycoprotein can also alter T-cell migration into the lungs (Figure 2) [99, 100]. The amino acid sequence of this glycoprotein contains an immune system-related CX3C chemokine domain that modulates T-cell migration and homing to the lungs. It is thought that this domain binds to CX3CR1 expressed on T cells [101, 102]. Indeed, the CX3C motif of the RSV G protein has been suggested to be responsible for reducing the frequency of CX3CR1+CD4+ and CX3CR1+CD8+ T cells in the lungs of infected animals, as well as the frequency of IFN-γ-secreting CX3CR1+ RSV-specific T cells in this tissue (Figure 2) [102]. This process is likely to be mediated by the soluble form of the RSV G protein diffusing from the site of infection (Figure 1) [103].

THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES

Current available therapies for respiratory syncytial virus

A strategy that has been approved and made commercially available to prevent RSV infection consists of the intravenous administration of a neutralizing humanized monoclonal antibody that inhibits the fusion capacity of the RSV F protein [104]. This approach is aimed at decreasing the probability of RSV infection, mainly in preterm and other susceptible infants. Along these lines are studies in mice showing that targeting the conserved domain within the RSV G protein, which harbours a CX3C-associated motif with monoclonal antibodies, can reduce both inflammation and virus titres in the lungs [18, 105, 106]. Consistent with this notion, these anti-RSV G protein antibodies are produced poorly after natural infection with RSV [16-18]. Combined with anti-RSV F neutralizing antibodies, monoclonal antibodies directed to the CX3C portion of the G protein could provide a more powerful method for preventing and treating RSV-associated pathology [18, 104]. Although monoclonal antibodies have been proven to be effective at reducing RSV infection, it is still somewhat controversial whether antibodies generated against this virus during natural infection protect or not [14-17]. In any case, antibodies directed against RSV are likely to form immune complexes with the virus, which may be captured by Fcγ receptors expressed on the surface of DCs and presented to T cells [16, 17, 107-110]. A recent report showed that RSV–antibody immune complexes captured by Fcγ receptors on the surface of murine and human DCs can modulate the activation of CD4+ and CD8+ T cells against RSV [27]. Furthermore, it was observed that the ratio of murine CD4+/CD8+ T cells, which may be linked to adequate antiviral immunity, depends on whether these antibodies are neutralizing or not [27]. These studies will contribute to understanding the relationship between antiviral antibodies and T cells, a question that has been poorly assessed for viruses in general.

On the other hand, replication of RSV in the lungs may be treated with ribavirin, an analogue of purine nucleotides. Ribavirin is thought to interfere with virus dissemination by promoting disruptive mutations [7]. However, clinical trials aimed at assessing the use of ribavirin against RSV have ultimately displayed insufficient power for determining whether RSV treatment with this drug is effective or cost-effective, so the drug is approved for use in preterm infants experiencing severe infection only in a few countries [7]. A newly developed strategy directed at inhibiting viral replication in the lungs is the administration of small interfering RNAs directed against RSV genes, such as the phosphoprotein gene P and nucleocapsid N for their degradation [111-113]. This later approach is currently under clinical evaluation [111].

Failure of an inactivated respiratory syncytial virus vaccine

Only a few years after the virus was identified, a formalin-inactivated RSV preparation (FI-RSV) was used in a field trial during the mid-1960s as an initial attempt to vaccinate against the virus [114]. Unfortunately, after natural infection with RSV, children that had received this formulation showed an exacerbated pulmonary disease and suffered more severe symptoms than unvaccinated children [114, 115]. Data explaining the failure of FI-RSV as a vaccine were obtained decades after intense research and suggested that the formalin-inactivated RSV administered prior to infection with the virus promoted immune polarization to an allergic-like response in the lungs, causing exacerbated disease [116]. In animal models of RSV infection, immunization with inactivated virus followed by challenge with infective RSV also induces massive eosinophil infiltration to the lungs [116-120]. In addition, immune complex deposition and complement activation has been observed in both mouse models and infected patients [117]. This is due to the establishment of unbalanced Th1–Th2 polarized responses, which are characterized by the secretion of pro-inflammatory cytokines that drive excessive infiltration of eosinophils and neutrophils into the lungs. Although modification of viral epitopes by formalin was initially thought to be responsible for the inadequate immune response displayed after immunization with FI-RSV [121], the development of low-affinity antibodies against this inactivated virus was recently suggested to contribute to the observed damaging immune response by impairing virus neutralization and thus efficient viral clearance [119].

Current vaccine development to prevent respiratory syncytial virus-induced damage

Although a few passive prophylactic strategies are currently available for preventing infection of high-risk groups with RSV, cost-effective vaccines are highly demanded by public health systems to reduce hospitalization rates and the economic burden produced by this virus [5]. After the FI-RSV failed to produce immune protection, several new strategies aiming at inducing active prophylaxis against RSV infection were tried [114]. Thus, a formulation conferring protective immunity against RSV with minimal immunopathogenesis has been the goal of researchers around the world. However, to date no vaccine strategy capable of inducing active and long-lasting immunity to prevent RSV infection has reached the public. Given that the immune response against RSV is pivotal in the pathology to this virus, prophylactic treatments aiming to modulate this response in neonates are likely to be the best approach for developing an efficient vaccine. Such a vaccine should prevent pro-inflammatory T-cell polarization induced by RSV and overcome inhibition of naïve virus-specific T cells upon infection. This can be achieved by activating T cells with DCs induced to secrete balanced Th1/Th2-polarizing cytokines that promote an efficient immune response against the virus. A traditional strategy that has worked for several pathogenic viruses and bacteria involves the development of attenuated strains displaying minimal pathogenicity that preserve sufficient immunogenicity. Such attenuated strains have the advantage of expressing most of the pathogen's antigens, while maintaining virulence traits from the parental strain, strong enough to alert the immune system [122, 123]. Using this strategy, attenuated RSV strains have been developed that are fully restricted for growth at body temperature and thus are safe [122]. Other RSV strains have been engineered to carry gene deletions or point mutations at key viral genes that encode immune-modulating regions, such as the attachment glycoprotein G [123]. Another approach involves the generation of recombinant RSV strains carrying host cytokines that promote Th1-type immune responses, such as IL-2 or GM-CSF, as a mechanism to deliver activating cytokines to the site of infection [90, 91]. Similarly, RSV genes have been engineered to be expressed as chimaeras in other viruses, such as vaccinia virus, Sendai virus, parainfluenza and adenoviruses, which is a novel strategy for delivering viral antigens against RSV [92, 124]. However, a possible disadvantage of these approaches is that they may display reduced safety or lack sufficient immunogenicity to establish protective immunity against RSV [124-126].

Alternatively, formulations consisting of recombinant protein subunits have been increasingly used for vaccine development. These formulations can be applied either alone or in combination with bacterium-derived or plant-derived adjuvants to increase viral antigen immunogenicity [89, 127]. Although this approach benefits from increased safety, they may show reduced immunogenicity or unbalanced immune polarization [99, 100, 128], so they have not been developed further into clinical phases [125].

Another approach that requires further evaluation is the use of recombinant attenuated bacteria expressing RSV antigens [21, 129-131]. Because bacteria express many pathogen-associated molecular patterns on their surface, they can induce strong Th1, Th2 or Th17 polarizing immune responses directed to the recombinant heterologous antigens that they express. Recently, our group has generated strains of Bacille Calmette Guérin (BCG, an attenuated strain of Mycobacterium bovis) expressing either the N or M2-1 RSV antigens (rBCG-RSV) that induce an immune response that protects against virus infection in a mouse model [21, 28]. Unlike other strategies, this approach intends mainly to produce virus-specific T cells and not antibodies to the virus. We observed that rBCG-RSV produced RSV-specific protective Th1 immunity characterized by IFN-γ-secreting T cells and simultaneously prevented the generation of T cells producing Th2-type cytokines [28]. Another advantage of this formulation is the capacity to prevent inflammation or cellular infiltration in the infected lungs [21]. Furthermore, rBCG-RSV-induced T-cell immunity led to undetectable viral loads in the airways of challenged animals [28]. These data suggest that T cells induced after rBCG-RSV immunization can confer immune protection against RSV and that these cells are refractory to the mechanisms used by the virus to downmodulate adaptive immunity [21]. Because BCG has been used worldwide for nearly a century as a routine vaccine against tuberculosis (more than four billion doses administered to humans since 1921, http://www.who.int/vaccine_research/diseases/ari/en/index4.html), with a good safety record in newborns, it is possible that this approach might lead to an affordable and efficient vaccine against RSV [21, 130].

Another study has used an attenuated Salmonella strain as a bacterial vector for the delivery of RSV antigens, which induces immunity in an animal model of infection [129]. Oral administration of this vaccine promoted a balanced Th1/Th2 immune response characterized by the expansion of RSV-specific antibodies and cytotoxic T cells, reducing lung viral loads in infected mice [129]. Although there are no data showing how such vaccines can affect DC function, the induction of protective immunity by recombinant bacteria expressing RSV antigens suggests that these formulations can modulate the immune response to produce virus-specific IFN-γ secreting T cells and thus should be considered as potentially affordable and effective alternatives for immunizing against RSV.

These animal immunization studies have provided compelling evidence for the feasibility of prophylactic priming with immunogenic formulations prior to RSV exposure that can elicit protective immunity capable of preventing infection and pathology by the virus [21, 89, 90, 92, 127, 132]. Moreover, even if RSV virulence mechanisms are aimed at interfering with the establishment of an effective primary immune response, if virus-specific T cells are efficiently activated and differentiated by adequately primed DCs prior to natural infection, they may then be able to override inhibiting signals provided by virus-infected cells. This provides hope for the development of new effective vaccines against this widely disseminated pathogen.

CONCLUDING REMARKS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES

Research throughout these past years has defined RSV as a virus capable of producing excessive inflammation in the lungs while simultaneously interfering with T-cell function by modulating the immunobiology of DCs. However, severe disease leading to hospitalization after RSV infection only occurs in a fraction of individuals who are particularly susceptible to developing severe lung pathology. Ultimately, this process may lead to the establishment of an immunopathogenic-like disease. The identification of host and viral determinants defining this higher susceptibility and severity of disease after infection remains elusive. However, host genetic factors are likely to play relevant roles during infection and, thus, identifying and associating these markers with increased susceptibility is fundamental for the design of useful prognosis kits intended to predict disease outcome. On the other hand, new immune components have recently been shown to participate during RSV-induced pathology, which could help to define what differentiates an efficient viral immune response from a pathologic one. In this regard, alteration of DC function by RSV is likely to be a key event at determining the nature and function of the T-cell response during infection. Accordingly, RSV has developed molecular strategies to interfere with the capacity of DCs to efficiently activate T cells, such as blocking DC–T cell IS assembly, although the mechanism by which this occurs is currently unknown. Furthermore, whether increased susceptibility to suffer severe pathology is mainly attributable to the DC–RSV interaction remains to be assessed. T-cell commitment after interaction with DCs will be defined in part by surrounding cytokines provided by the innate immune response. However, the question as to how these factors trigger detrimental T-cell immune responses, particularly in susceptible individuals, remains unanswered. Identifying the mechanisms by which these cytokines influence the outcome of T cells is crucial, as well as the signalling events taking place in T cells primed by RSV-infected cells.

Addressing these questions should shed light on the mechanisms used by the virus to impair T-cell function, providing tools for predicting the severity of RSV-induced pathology and contributing to the design of effective and safe vaccines that induce balanced and robust protective immunity against RSV.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES

The authors are supported by grants FONDECYT nº 1070352, FONDECYT nº 1085281, FONDECYT nº 1100926, FONDECYT nº 3070018, FONDECYT nº 3100090, FONDECYT nº 11075060, FONDECYT nº 1100926, FONDEF D06I1008, SavinMuco-Path-INCO-CT-2006-032296 and Millennium Institute on Immunology and Immunotherapy. A. M. K. is a Chercheur Étranger D'Excellence, Chaire de la Région Pays de la Loire.

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  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. RESPIRATORY SYNCYTIAL VIRUS IMMUNITY
  5. THERAPY AND PROPHYLAXIS FOR RESPIRATORY SYNCYTIAL VIRUS
  6. CONCLUDING REMARKS
  7. CONFLICT OF INTEREST
  8. ACKNOWLEDGEMENTS
  9. REFERENCES
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